IFN-γ ELISPOT Assay

CD4+ T cells were depleted from PBMCs by using anti-CD4 MAb-coated magnetic beads (Biolegend, San Diego, California, USA) as described in the manufacturer's instructions. The Human IFN-γ ELISpot Kit (Mabtech) was used to detect secretion of IFN-γ according to the instruction manual. 2 × 10⁵ CD4+T cell depleted PBMCs were added per well in duplicate in a volume of 200 μl, and the HIV-1 gag peptide pools or CEF peptide pools (20 μg/ml, ProImmune) were added for 20 h. Anti-CD3/CD28 beads (3 μg/ml) was used as a positive control, and negative controls consisted of cells without stimuli. Analysis was done using the ImmunoSpot plate reader.